Summary/Abstract (Project 2) Anti-factor VIII (fVIII) alloantibodies (inhibitors), which can develop in patients with hemophilia A, limit the therapeutic options for these patients, and can increase morbidity and mortality. Unlike most immunogens encountered by a host, fVIII possesses no canonical innate immune ligands known to activate host immunity. Despite this, nearly 30% of patients develop inhibitors following fVIII exposure. The inability to prevent inhibitor formation largely stems from a fundamental lack of understanding regarding key features of fVIII that are responsible for engaging and then activating host immunity. Our long-term goal is to define key features of fVIII that initiate and then enhance inhibitor formation following subsequent fVIII exposures, in order to reduce or prevent inhibitor development. Our central hypothesis is that key glycan signatures on fVIII facilitate initial host recognition and response to fVIII, followed by the formation of anti-fVIII specific antibodies that in turn impact the immunological outcome of subsequent fVIII exposures. Our hypothesis is formulated based on our recent discovery that different fVIII products not only possess unique glycan signatures and distinct abilities to induce inhibitors, but also that fVIII engagement by distinct anti-fVIII antibodies differentially impacts host immune cell interactions. Thus, fVIII glycans and early anti-fVIII antibodies may represent key early regulators that dictate the ultimate immune outcome of fVIII exposure. As unique glycan structures can differentially engage distinct innate immune receptors, these results strongly suggest that individual fVIII glycoforms may differentially impact host recognition and inhibitor development. Furthermore, the ability of distinct anti-fVIII antibodies to differentially impact fVIII uptake by antigen presenting cells strongly suggests that the nature and epitope specificity of early anti-fVIII antibodies likely shape the immunological consequence of subsequent fVIII exposure. Finally, as non-human glycan epitopes present on certain fVIII products can be recognized by naturally occurring anti-?Gal antibodies, anti-?Gal antibodies (stimulated by distinct microflora) may likewise bind fVIII and impact the likelihood of inhibitor formation. We will use a complementary approach of biochemical analyses and in vivo studies to define key features of fVIII responsible for inhibitor formation by testing the following specific aims: Aim 1: Define the role of fVIII glycans on inhibitor formation. Aim 2: Define the role of anti-fVIII antibodies on fVIII immune complex formation and inhibitor development. Aim 3: Define the impact of microbiota on anti-?Gal antibody development and subsequent inhibitor formation. We believe that successful completion of these aims not only possesses the capacity to define key features of fVIII that regulate inhibitor formation, but may also establish an important framework to develop rational approaches designed to develop unique fVIII products with reduced immunogenicity.